Identifying targets of the Piwi/piRNA pathway in stem cells

描述（由申请人提供）：需要成体干细胞来维持组织稳态，干细胞功能的下降会导致与衰老相关的健康下降。干细胞衰老的原因尚不清楚，探索干细胞调控的基本机制对解决这一问题至关重要。简单的后生动物Hydra vulgaris显然是“不朽的”，而该属的其他成员则经历了繁殖引起的衰老。比较来自两种具有完全不同衰老策略的密切相关动物的干细胞调控的分子机制的能力是一项独特的研究

DESCRIPTION (provided by applicant): Adult stem cells are required to maintain tissue homeostasis and decrease in stem cell function leads to the decline in health associated with aging. The causes of stem cell aging are poorly understood, and exploring basic mechanisms of stem cell regulation is vital to address this issue. The simple metazoan Hydra vulgaris is apparently "immortal", while other members of the genus undergo reproduction-induced senescence. The ability to compare the molecular mechanisms underlying the regulation of stem cells from two closely related animals with starkly different aging strategies is a unique and attractive feature of Hydra for aging studies; other well-established model organisms do not have seemingly "immortal" relatives. Furthermore, Hydra stem cells have attractive features for experimentation including: 1) Stem cell populations are well-characterized, 2) Due to recent advances in transgenic methods, the stem cells can be easily collected in large numbers and can be tracked and manipulated in vivo, and 3) Hydra stem cells share a gene expression profile with stem cells found in more complex animals. This includes the Piwi proteins, which are required for germline and stem cell function in animals. Our preliminary data suggest that Hydra Piwi proteins operate in a stem cell-specific pathway for maintaining tissue homeostasis and epithelial integrity in response to nutritional status. Because nutritional status is a conserved regulator of lifespan, we hypothesize that the Piwi pathway is a key player in the aging pathway of stem cells and we aim to understand how it functions in this context. Piwi proteins associate with a distinct class of small RNAs, piRNAs, and in Drosophila and mouse these Piwi/piRNA complexes modulate gene expression at the epigenetic and post-transcriptional levels. Hydra vulgaris stem cells have two strictly cytoplasmic Piwi proteins, allowing us to focus on identifyin post-transcriptional targets of the pathway. We are using a transgenic approaches to knockdown Piwi protein expression and test the function of the Piwi/piRNA pathway in each Hydra stem cell type. To identify targets of the pathway, we are: 1) Mapping piRNAs to a Hydra transcriptome to identify putative targets, 2) Testing for increased transcript levels of putative targets in piwi knockdown animals, and 3) Testing for a physical interaction between the putative transcript and Piwi/piRNA complexes. Finally, we are testing the hypothesis that the Piwi/piRNA pathway responds to nutritional status through the insulin pathway to regulate stem cell proliferation and tissue homeostasis. We aim to understand both the upstream regulators of the pathway and the downstream targets. Ultimately, we hope to gain significant insights into the regulation of aging in stem cells that will translate to more complex organisms. My long-term goal is to establish Hydra as an important new model for dissecting the regulatory control of stem cell function and aging. Once I have established my own lab I will initiate collaborations in order to test the conservation of the Piwi/piRNA pathway in stem cell regulation in vertebrates.